In the coming years, our goal is to understand how motile microbes, such as P. aeruginosa, regulate the transition to a biofilm lifestyle. A key aspect of this transition is the regulation (perhaps ?modulation? is a better word) of the three surface behaviors of this microbe: flagellar mediated swarming, TFP-mediated twitching motility and EPS-dependent biofilm formation. The experiments proposed below seek to understand the mechanism(s) underlying the coordination of these surface behaviors. An integral aspect of this work is defining how this microbe detects surface engagement; our work on PilY1 is allowing us to dissect one such ?surface sensing? pathway. My group has already made contributions to this exciting area of microbiology, and we will continue to do so in the come funding period by (i) completing studies proposed in the original application and (ii) breaking open new areas of research. To address the latter, I propose to incorporate a new formal and funded collaboration into years 6?10 of the project with Dr. Gerard Wong at UCLA. We have already co-published five papers with a 6th paper in press at PNAS. We propose the following studies to continue to test the central hypothesis that cdG levels are up-regulated in response to cell-to-substratum contact, leading to reduced motility and promotion of biofilm formation. Aim 1. Test the hypothesis that a protein-protein interaction cascade signals from PilY1 to SadC to modulate c-di-GMP levels. Aim 2. Test the hypothesis that SadC coordinates swarming and twitching motility via inner membrane complexes. Aim 3. Test the hypothesis that stator swapping drives distinct behaviors during early attachment. Aim 4. Test the hypothesis that modulation of flagellar- and TFP-mediated motility contributes to the formation of polymicrobial communities. Aim 5. Test the hypothesis that c-di-GMP signaling is required to maintain mature biofilms. RELEVANCE (See instructions): Bacterial biofilm-based infections are estimated to cause upwards of a billion dollars annually in increased medical costs. The work we propose here will reveal mechanisms whereby these communities form, and thus provide insight into how to prevent such infections.